DESCRIPTION (provided by investigator): HIV-1 associated nephropathy (HIVAN) is a disease largely limited to individuals of African descent. Although its pathogenesis is not completely understood, it is known that expression of HIV-1 genes, interacting with the individual's genetics, is required for HIVAN. Genome-wide studies recently identified MYH9 as a candidate gene associated with the risk for development of HIV-associated focal segmental glomerulosclerosis (FSGS) in African Americans. Although the mechanism of kidney infection remains undetermined, HIV-1 nucleic acids and proteins have been detected in podocytes of patients diagnosed with HIVAN. Since renal biopsies are not usually performed on HIV-1 infected patients without kidney disease, it is uncertain whether HIV-1 can infect renal epithelial cells in patients without HIVAN. Critical to the blood filtration barrier, podocytes lost into urine as a result of HIV-1 infection may promote HIV-associated FSGS. Previous studies and our preliminary data show that urine is a source of viable human podocytes. Podocytes isolated from the urine of HIVAN patients can help us clarify HIVAN pathogenesis including the mechanism(s) leading to their infection. Podocytes lack classical HIV-1 entry receptors CD4, CXCR4, or CCR5 so virus entry through this pathway is unlikely. We show that HIV-1 readily enters podocytes through an endocytotic process leading to unintegrated HIV-1 DNA that does not express HIV-1 structural proteins. In vivo detection of HIV-1 nucleic acids and proteins in HIVAN podocytes may not reflect productive replication but rather that HIV-1 endocytosis leads to a limited reverse transcription of the viral genome without integration. Transcriptional activity of unintegrated HIV- 1 DNA has been shown to direct the expression of spliced viral transcripts and synthesis of HIV-1 accessory proteins, including Nef. Nef is one of the earliest and most abundantly expressed viral proteins proposed to damage podocytes in HIVAN. We have shown that Nef is released in vitro by HIV-1 infected podocytes in association with exosomes. Nef can also be found in HIV-1 particles, however, only a few HIV-1 particles escape into the cytoplasm where they can release Nef during uncoating. Thus, dissemination of Nef within exosomes could spread damage to uninfected podocytes without productive replication. We propose that HIV-1 entry into podocytes results in an abortive infection plus the secretion of exosomes enriched in Nef that in turn dysregulate uninfected podocytes leading to HIVAN. To test this hypothesis, we propose 1) to investigate the mechanism of HIV-1 infection of podocytes isolated from the urine of normal donors and HIV+ patients without and with HIVAN via fusion assay, Western blotting, confocal microscopy and real-time PCR, and 2) to examine the potential contribution of exosomes secreted by HIV-1 infected podocytes and encapsidating Nef in the promotion of HIVAN-specific damage in uninfected podocytes via analysis of changes in the cytoskeleton, adhesion and slit diaphragm components as well as expression of pro-apoptotic factors in uninfected podocytes exposed to Nef-containing exosomes. PUBLIC HEALTH RELEVANCE: HIV-related kidney disease (HIV associated nephropathy, or HIVAN) is almost entirely limited to people of African descent, with over 90% of HIVAN patients being black. In the laboratory, HIV does not easily infect kidney cells, so this project seeks to discover if HIVAN is the direct result of HIV infecting the kidneys, or if the effect is due to some other action of the virus or the body's reaction to it. Understanding the interaction of the virus with specific kidney cells, called podocytes, may help us develop treatments or early assessments of the risk for developing HIVAN in people of African descent.